Failure to detect antibody against Gross virus in tetraparental AKR reversible CBA mouse chimaeras.

In spite of early acquisition upon the germ line, tolerance to the Gross (gs) virus is short-lived in the AKR. From about the age of 3 months anti-gs antibodies occur and these complex with the corresponding viral antigens. Such complexes are best seen in the glomeruli by means of immunofluorescence. In marked contrast to the AKR, renal complexes were minimal in a group of AKR reversible CBA/H-T6 chimaeras derived by early embryo aggregation. This was particularly surprising since large numbers of type C murine leukaemia virus-like particles were identified in the chimaeras and the tissues were found to be saturated with gs antigen. The lack of renal antigen-antibody complexes was the first suggestion that anti-gs antibody might not be present in the chimaeras and renal elution studies here support this assumption. In contrast to the AKR where "split " renal eluates have been shown to have anti-gs activity, no activity was demonstrated in eluates from any of the chimaras. Tolerance to the oncogenic Gross virus in the chimaeras has to be attributed to the CBA parental strain component and since this component is also held responsible for the tumour resistance of these chimaeras, both phenomena could well be related. In this context it would appear that in the absence of masking by antibody viral antigenic complexes, tumour specific sites can be recognized in the chimaeras and unlike the AKR "normal" tumour immunity can be effected. This hypothesis is currently bei-ng tested.

THE AKR strain of mouse is characterized by the development of " spontaneous" lymphomata (Furth, Seibold and Rathbone, 1933). " Gross " classic experiments (Gross, 1951) established the role of the oncogenic virus and it is generally assumed that this is acquired in the AKR upon either germ line. In spite of early infection, tolerance to the Gross (gs) virus in the AKR is not permanent, since both Markham and his colleagues (Markham et al., 1972) and Oldstone (Oldstone, Aoki and Dixon, 1972) demonstrated anti-gs antibody activity in renal eluates prepared from the AKR. Since anti-gs antibody activity occurs at a time when lymphomata first develop, we recently suggested that these two events are probably related (Barnes,1

974a).
Susceptibility to virus associated mu-I rine tumours is dependent upon genetically determined host factors. The H-2k and Fv-ln loci are associated with virus induced tumour susceptibility and oncogenic viral replication respectively (Lilly and Pincus, 1973) and it is not surprising to find that the AKR are positive at both of these loci. Curiously enough, this is also true for the CBA which are generally resistant to lymnphomata (Murphy, 1966). Results obtained in an embryo transfer experiment led us to suggest that there was an additional and dominant factor responsible for the lymphoma resistance of the CBA. Even after transplantation at the early blastocyst stage and being born from the AKR, the CBA still remain lymphoma resistant . In the reverse experiment, the fact that AKR derived from the CBA still developed lymphomata arguied that any maternal CBA effect beyond the stage of implaintation was of no consequence in respect of influiencing the inniate lymphoma susceptibility of the AKR (Barnes and Tuffrey, 1 974b). To rtudy the possible interaction between f actors associated with tumour stusceptibility in the AKR and those leadinig to tumniotur resistanice in the CBA, tetraparental AKRI-*CBA/H-T6 chimaeras wrere subsequently investigatedl.
These were derived by aggregation of early emnbryos Usinlg the techniques described by Tarkowski (1961) and Mintz (19(62). Initial findings in a group of 18 tetraparenital AKR-CBA/H-T6 clhimaeras showedl that on conmparison with the AKR the incidence of lymphomata was both delayed (Barnes, Tuffrey and Kingman, 1972a) and also reduced . Tuimour resistaince in this situation coul(d not be explained by absence of lymphoma-prone AKR cells since cytogenetic analysis of both peripheral blood ctultures  and other tisstues (Ford et al., 1974) showed an overwhelming preponderance (>900 %) of AKR cells.
Tuimour resistaince of the chimaeras also cotuld not be explained by the absence of the oncogenic virtus. Large numbers of type (C murine leukaemia virtus-like particles were seen on electron mnicroscopy (W!ills, Tuffrey and Barnes, uinpublished) andl Gross (gs) anitigenic specificity was subsequently confirmed . In both cases the findings were comparable with the AKR. However, in contrast to the AKR (Oldstone et (Barnes et al., 1972b), together with earlier results (Barnes et al., 1972a;Barnes, 1974a;Bona, Tuffrey and Barnes, 1974;Ford et at., 1974) have been described previously. To facilitate comparison, the original reference numbers lhave been retained here. The ehimaeras were examined together with both AKR/J and CBA/H-T6 (CBA in text) controls.
2. Inrvestig(ation. Portions of kidney and thymus wN-ere removed from both the chimaeras and the controls. These were snap firozen in isopentane in an equilibrated isopentane-solid CO2 mixture and stored at -35°C until investigated. Portions of the kidney were homogenized in a Potter inill. Renal eluates were subsequently prepared and " split " to release antibody according to the technique of Oldstone et at. (1972). In the case of the AKR and CBA controls, the renal eluates were subsequently pooled each to represent groups of 3 mice. All eluates were subsequently dialysed and concentrated by perevaporation. After initial testing, eluates prepared from the chimaeras were fitially pooled together, concentrated further and then re-tested as described below.
Aliquots of eachi eluate wNere applied to cryostat cut unfixed AKR and CBA thymus tissue sections. Following incubation (20TC, 20 min) and washing, the section was then treated with fluorescein labelled goat antimouse Ig (20TC, 20 min). This conjugate was used at a prior determined optimum dilution with 10°, lissamine rhodamine conjugated BSA as a counterstain.
In an attempt to block subsequent reactivity of the renal eluates against gs-antigenic sites, certain sections were first treated with specific goat anti-MuLV-gs antiserum. The antiserum, kindly provided by Dr R. G. Gilden, was prepared against isoelectrically purified gs-antigen and details concerning its preparation and specificity have been described previously (Oroszlan et at., 1970). Following incubation (20°C, 20 mnin) with a 1: 50 dilution of this antiserum and washing, the section was then treated as before with the renal eluate.
Thlis was follhwed, after washing, by incuba-tioui with fluoresceimn labelled a All sections were subsequentl) buffered glycerol and examinc Orthoplan microscope fittedincident light attachment. TI scored upon a double-blind basi ing " controls -were included.

RESULTS
'rfhe resuilts are suimmna Table, where it cani be sec AKR renal eluates showed a against the thymus. As ( from the  rThis stainiing appeared sOme than that originally (lescribe' (Oldstone et al., 1972) tions. We recently proposed that the O1 ( 9 clevelopment of anti-gs viral antibodies in the AKR might also be associatedl with clinical manifestations--namelv the from4 grI)ts of lvmphoma of the AKR (Barnies, 1974b).
involving, i in this conitext, we suggested that antite followed( by bodv viral aiitigenic complexes might egoet anti-effectively mask " tuimouir specific sites in. the AKR and in this situation " nornmal'' tn.mour immunie mechanisms couild :what weaker not be effected. This suggestion. was I by(Oldstone largely based upon evidence obtained staining was from the tetraparental AKRlC(BA/H-T6p; or the chi-chimaeras. ling evenl the As mentionie(d earlier, the tumlnouir I chimacera resist--ance of the AKR-('BA/H-i'6 chimllaeras has to be attributetd to the ('BA ltv in inter-p)arental strain. componienit. In. spito of e AKR renal a 100% inci(lence of lmphomata by issue sections the a(re of 56 wveelks in the AKR (Blarnies gs antiserum anid TuifreyNI, 1 97 4b) the majority (660o) ,taining, thus of the AKR"C-BA/H-T6 chimaeras lived for tip to more thaii three times the II average life span of the AKR  and, furthermore, when examined histologically were found to be free of lymphomata (Barnes, Tuffrey and Wills, unpublished).
Tumour resistance of the chimaeras could neither be attributed to absence of AKR cells Ford et al., 1974), AKR cell products (Barnes et al., 1974a) nor the oncogenic Gross virus Wills et atl., unpublished). Tumour resistance therefore had to be attributed to another factor.
The H-2k and Fv-ln loci are known to be associated with virus induced murine tumour susceptibility and replication of oncogenic viruses (Lilly and Pincus, 1973). In retrospect, it was surprising to note that like the AKR the CBA are positive at these loci but in spite of this the CBA are resistant to lymphomata (Murphy, 1966). Even after transplantation at the early blastocyst stage and being born from the AKR, the CBA still remain resistant to lymphomata . It was this fact that led us to postulate the presence of another and furthermore dominant factor responsible for lymphoma resistance of the CBA. It now appears that this factor not only overcomes tumour susceptibility associated with the H-2k and Fv-ln loci in the CBA, but also overcomes the innate AKR susceptibility in the AKR-CBA/H-T6 chimaeras.
The fact that in spite of transplantation at the early blastocyst stage and being born from the CBA, the AKR still develop lymphomata, argues that the " cause " has to be established before implantation . Maternal influence beyond this stage is of no consequence in respect of tumour susceptibility; however, incorporation of CBA cells into the AKR during early embryonic life has obviously conferred an advantage in respect of tumour resistance in the AKR-CBA/H-T6 chimaeras.
In striking contrast to the AKR (Oldstone et al., 1972) renal antibody-antigenic complex staining was minimal in the chimaeras . This difference was particularly remarkable since the chimaeras were predominantly AKR in both cellular Ford et al., 1974) and cellular product composition including the antibody associated serum allotype (Barnes et al., 1974a). Apart from being predominantly AKR, and in spite of the fact that the chimaeras were saturated with gsantigen , renal complex staining was minimal. This was the first suggestion that anti-gs antibody might not be present in the chimaeras. This view is supported by the findings here.
Although weak positive staining of AKR thymus sections was seen with AKR renal eluates, this was not invariable. However, it now appears that positive staining can be demonstrated only in certain renal eluates and these prepared from the AKR with the most marked renal lesions (Oldstone, personal communication). Although the staining observed with the AKR renal eluates was very much less convincing than that earlier described by Oldstone (Oldstone et al., 1972) there were clear differences between the staining observed with AKR renal eluates and the invariable negative staining seen with both CBA and AKR-CBA/H-T6 renal eluates. In spite of relatively weak staining with the AKR renal eluate, anti-gs specificity was confirmed by successful blocking by prior treatment of the AKR thvmus substrate section with the specific anti-MuLV-gs sera.
Absence of staining with chimaera eluates can be explaiined in one of two ways. Either antibody was present but in insufficient amounts for detection or anti-gs antibody was not present. The latter view seems more likely since reactivity could not even be demonstrated with the highly concentrated pooled chimaera renal eluate. More important was that treatment of an AKR thymus tissue section with this eluate failed to " block " reactions with both a known positive AKR renal eluate and also the specific goat anti-MuLV-gs serum (Barnes and Holliday, unpublished data). Evidence therefore favours the absence of anti-gs activity in the chimaeras. This has to be attributed to relatively few CBA cells or their products and there is a clue how this has been achieved. Cytogenetic analysis of PHA stimulated peripheral blood cultures showed an overwhelming predominance of AKR cells (>99%) . In contrast analysis upon the basis of 0 antigenic surface determinants showed a roughly balanced admixture of AKRAKRO and CBACBAO cells (Bona et al., 1974). These results initially appeared paradoxical since it is the T cell that is stimulated by PHA and it is this cell that has 6 antigenic determinants. Recent results appear to have resolved the paradox. Cytogenetic analysis following treatment with anti-AKRO in the presence of complement and subsequent T cell stimulation (Con. A) revealed a significant number of AKR cells (Barnes, Tuffrey, Bona, Evans and Ford, unpublished data). This led us to consider the existence of AKR T cells without their corresponding AKRO antigen and the possibility that these cells might be processed with CBA0 antigen. If proven, this suggests that 6 processing would have occurred independent of the cellular genome. In man the Lewis red cell group is acquired from a source other than the red cell or its precursor (Race and Sanger, 1958). It is conceivable that a similar process has occurred here in respect of 0 processing.
Theta processing might well be a vital clue in respect of tumour immunity. In contrast to most AKR sublines, two sublines, namely AKR/Cum and AKR/ RuA appear exceptional in not only being relatively resistant to lymphomata but also in having QC3H rather than QAKR (Acton et al., 1973). Conceivably these two factors might be related and thus we are left with the possibility that CBA 6 processing of AKR cells in the chimaeras may have been directly responsible for their tumour resistance. It is an attractive hypothesis to consider that this process of tolerance to the oncogenic Gross virus, may have been maintained and in the absence of " masking " of tumour specific sites by antibody-viral antigen complexes " normal " tumour immune mechanisms are effected. This hypothesis, however, remains to be proven.
We are grateful for the expert advice of Dr Jo Hilgers and Dr June East. The expert technical assistance of Misses Jean Kingman, Pamela Lund and Linda Dawson is acknowledged.